Lepr Rev (1998) 69 128-133

Choosing the decolourizer and its strength to stain Mycobacterium leprae Does it actually matter

R DE SOLDENHOFF M HATTA amp T WELING SIROmiddot NSL-South Sulawesi Leprosy Control Project Provincial Health Services Sollth Sulawesi Indonesia Departll1ent of Microbiology Medical Facillty of JuWllIddin University Ujwlg Pandang Indolles([

Accepted for pubhcation 15 April 1998

SIIIIIIary Leprosy bacilli are more easily decolourized during staining than tuberculosis bacilli so a weaker concentratIOn of decolourizer is usually recomshymended In Indonesia the same strong decolourizer is used for identifying both urganisms Tn a study to compare the results llsing different concentrations of different decolourizcrs no differcnce could be found in thc bacterial index (HI) It is suggested that the same staining technique can be used for tuberculosis and leprosy

Introduction

Ideally all leprosy patients should have one skin smear examination before starting treatment if reliable facilities are available Thc Ziehl-Neelscll method involves slaming with carbol fuchsin followed by decolourization then counterstaining with methylene blue If thc decolourization process is excessive bacilli may be rendered invisible Concerns regarding which deeolourizer to use what strength and for how long have resulted in several in vestigations 3-6

In Indo[]esia the same staining technique is normally usee for leprosy and tuberculosis despite the widely accepted opinion regarding the reduced acid and alcohol fastness of MycohacteriulIl leprae 2 The objective of this study was agaill to find out if (i) different decolourizers and (ii) different concentrations of the same decolourizer had an effect on the B I of skin sl1Icars

Although the Ziehl-Neelsen stain is used universally to stain vI leprae there are many modifications 7 The main difference between the technique for leprosy and tuberculosis is the strength of the decoourizer but in addition the duration of decolourizing varies For tuberculosis this is IJsually either 25 sulphuric acid or 3 hydrochloric acid in 70

Correspondence to NSL-Sulsel Leprosy lOl1lrol Project PO 130 10 11 lJlIng Panoang Sulawesi Indrlllesia e-mail richanis(J-ioupglllegalletid

128 0305-751898106) 118+06 S I (() roi leplltt

DecorJlrier Illd its strellgth ill the ZmiddotmiddotV stoill 129

ethanol for 3 min~ For leprosy decolourization or differentiation using I hydrochloric acid in 70 ethanol is favoured by l11ost27middot910 but 5 sulphuric acid by some I I Dharmendra

surprisingly recommends 3 acid aicohol12 The countcrstain is llsually mcthylene blue although the concentration varies between 0middot2 II and 16 Vettom and Pritze found 10 different techniques from 29 projccts the most notable variations being in decolourization time 13 They ranged from I acid alcohol for 5 s to 20 acid alcohol for I min and from 59(middot sulphuric acid for I min to 25 sulphuric acid for 10-20 min 11

Despite this several rcsearch laboratories use the same concentration of decolourler for leprosy as for tuberculosis (A McDougall personal communication) The Indonesian Icprosy manual describes two possible Ziehl-Neelsen methods one with acid alcohol (concentration not specified) for 3-5 s and the other with 25 sulphuric acid for R s Jj

Materials and methods

Skin smcars were takcn from 40 l11ultibacillary (MB) leprosy patients with a range of Bl The selection was by the technician who took the smears did the staining and rcad the results The patients were registered cases and were on WHO ivlB multidrug therapy (MDT) I All patients had smears taken from four standard sites with four identical slides being made from eClch patient each slide with the foul sites on it

Apart jrolll the decolourizcr the same Ziehl-Neelscn mcthod was used ls The decolourshyizers were Ik hydrochloric acid in 70k ethyl alcohol (HCI) 3ck hydrochloric acid in 70 ethyl Cllcohol 5k sulphuric acid (H2S04 ) and 25 sulphuric acid Slides were stained in batches CarboI fuchsin was filtcred onto the slide allowed to act for 2 min heated gently until steam rosc allowed to act for a furthcr 10 min washed with tap watcr until clean transfcrred to a staining rack and dipped in decolourizer for 8 s If any slide was still red this was repeated washed and counterstained with O3k methylene blue for I min Most slides only rcceived one nllIllersion ill the decolourizer

The intention was that the examination of the smears would be blind but it was possible to distinguish immediately between thc slides decolourized with HCl and those with H2S04 bull

by microscopy of the smear It was not possible to distinguish between I r and 39c HCI or between 5 and 25 H2S04 bull

The BI was reported for each of thc four smears on the slide and the avcrage was calculated The morphological index (MI) is not routincly used in Indonesia

The results were compared using the Students paired t-test](i Differences were considered signiflcant at the 95 level of confidence

Results

Forty paticnts had slit skin smears taken resulting in 160 slides Each slide had four smears except in the casc of patient 6 who fainted aftcr smears had been taken from two sites (ears) Thirty-four patients were positive with at least onc staining technique An average BI was cCllculated for each of the 160 slides (~ee Table I) Comparison was also made lIsing individual smcars

Eleven patients had a higher BI with 1 HCI than with 3 Twelve patients had a lower BI with 1 HClthan with 3 Eight patients had a higher BI with 5 H2SOj than with 25 Thirteen patients had a lower BI with 5 H 2SO1 than with 25

130 R de Sodell hoff et ai

When looking at low BIs (ie gt0 but lt2) the same pattern was obsened Elven patients had a low BI using 1 He In two cases the BI was greater using 1 and in five cases the BI was less Twelve patients had a low BI using 5 HgtSO- In three cases the BI was greater using 5 and in five cases the B1 was lower with 5

Tabl 1 BI r~suls for )(J patients using four decolourizers

Di n~reilce bel wcell Ic acid 3ltC acid Yk sulphuric 251lc sulphuic I (~ Hel amp 5bull

Patient alcohol alcohol Difference acid acid Difference sulphuric acid --~------~~- __-------------- - - - ----~--- - -------- - -_----_ - ----shy ---~-

17) 225 -05 2 1middot75 025 -0middot25 2 1 I 0 075 0middot75 [) 0middot25 3 )75 4middot5 (J25 - 425 middot025 [)75 4 1 I 0 I 0middot75 025 0 5 () () () Il 0 () 0 6 ) ) 0 3 ) I I 7 2 1middot75 ()25 I 125 --()middot25 8 3middot75 3middot5 0middot25 4 4 () -025 ) 45 375 075 4 4middot25 -0lt5 ()middotS

10 15 175 -025 125 125 () ()25 11 275 2middot5 (J25 175 2middot75 --I I 12 ()5 i middot5 -I 05 () 0middot5 0 13 1 175 -075 125 1middot25 () middot-025 14 0 (J 0 () 0 (J 0 IS 4middot5 4 0middot5 3middot2 3middot25 -15()

16 1middot75 2middot75 -I () 3middot25 -325 1middot75 17 25 275 -()25 () 25 -25 2middot5 18 125 I ()l) I () ()25 19 () () 0 () () IJ ()

20 -1middot25 -125 () 4~5 35 lmiddot75 0 21 ~75 225 (lmiddotS Imiddot 3middot5 -2 1~5 22 05 025 0 O~5 025()) l

23 () () 0 () 0 () (j

24 +middot75 +middot75 0 4middot5 4 (lmiddotS 025 25 () I) () () () 0 0 26 3middot~5 -1 -075 3middot25 -05 ()25 27 0 2middot25 -Ol5 0 () () 2 28 375 375 () 3middot75 375 (J 0 ~9 475 +75 () middotl~ 4-5 () Dmiddot25 30 35 375 -025 35 225 15 0 31 3middot5 ) -05 3 2middot5 0middot5 05 32 4 3middot75 ()25 -1middot25 425 0 -)25 33 5 0 5 5 () ()

34 125 OS 075 025 0middot75 -05 1 35 () (j 0 0 0 () 0 3( 35 -125 -0middot75 375 375 0 -025 37 2 35 -15 375 3middot75 () -175 38 0 125 -125 075 1middot25 -05 -075 39 0middot5 0middot5 () 0 125 -125 OJ 40 4middot75 -1middot25 ()middotS 475 4middot75 () II

Total 925 97 -45 7(j25 88middot25 -12 1625

Aerag~ 2middot31 243 (0 III IYI 2middot21 (()30) 0-41

SO 050 Ot) (J98

p value lt0middot2 lt(Jmiddotl ltU02

131 f)ec(I(lIri~er ([lid its strellgth ill the Z-N staill

The averagc of all thc Bfs using 1 HCI was 2middot31 (with seven negatives) The average HI using 1 HCl was 2A1 (with six negatives) The difference between the BIs wa~ not statistically significant (p lt 0middot2 and gt01) The average BI using 5 H2S04 was 1middot91 (with 11 negatives) Tlw average HI using 25r H2S04 was 2middot21 (with nine ncgatives) Again thc difference between the Bis is not statistically significant (p lt 01 and gtOmiddotOS) When the differences between the BIs ith 1 acid alcohol and 5 sulphuric acid were examined there was an average fall of 0middot41 This was signillcant at the 29c level (p lt 002) suggesting that there might be a real decrease in BI when using sulphuric acid

There was generally good correlation between the four slide results from each patient An exception was founli in patients 15 16 and 17 where the slides stained with 5(1 H2SO were negative whereas the other three were moderately or strongly positive The most likely explanation for this was that these slides were wiped clean on the wrong sick of the slide hence removing all four smears from the slide If the results from these three patients are excluded the average BIs arc 226 and 2middot36 for acid alcohol and 2middot06 and 214 for sulphuric acid The differences between these ligures (betwecn the two acid alcohols the two sulphuric aeids and between I( acid alcohol and Y( sulphuric acid) all fail to re1Ch statistical significance (Ii lt ()) I lt 0middot5 and IJ lt ()middotl respectively)

In 3g of 15~ sites cxamined there was a variation of two or morc BI units (ie Illore tlwn (I

1 O-fold diffelTnce in the number of bacilli) from the same site using differcnt dccolourizers No pattern was obscrved

Thc slides which were stained using an acid alcohol uecolourizer were generally easier to examine than those llsing sulphuric acic Irrespective of concentration the bacilli were sharper clearer more reel the background had accepted more blue for differcntiation and there was little or none of the background pink fuzz that is a feature of the slides which were decolourized llsing Slfc or 257lt H2SO

Discussion

POSITIVITY

This study failed to show that a stronger decolourize resulted in a lower gT The BI was slightly higher with the stronger concentration but not reaching statistical significance (see Figure 1) This was also noted when looking at the results from patients with a low laquo2) BI The Bl was slightly lower with sulphuric acid this reaching significance only when the questionable data from patients 15 16 and 17 were included

POSSIBLE BIAS

All patients were purposefully selected Nu attempt was made to havc a rcpresentative sample of leprosy patients new or otherwise Since the purpose of the study was to comparc results patient by patient analysis using an average BI for all patients per technique is open to question The difference in BI from the same site could also be affected by th~ quality of the smear and within-observer variation Three patients had results which suggest a major laboratory error Analysis was also performcd excluding results from these three patients

DOES IT REALL Y ~ATTER

Most new leprosy patients can be competently diagnosed and commenced on appropriate

132 R de SodellllOi) el ill

Patients

1--1 HCI +3 HCI +5 H2S04 ---25 H2S04l

Figure 1 Comparison of Bl results using four decolourizers

treatment without 1 skin smear However there are some patients who present with single or few lesions but who have early multibacillary disease 17 There are other patients either new or old who have no clearly demonstrable clinical cardinal signs but who have a positive smear There are suspicions that some multi bacillary patients who are released from fixed duration treatment but still with a BI of gt3 may have a significant relapse rate It has been suggsted that regular clinical and bacteriological review after release from treatment is indicated for these patients IS Where communications are difficult it may nut be practical to refer all smear examinations to a I eferral laboratory 19 Hence there is still be a need for skin smear examinatioll to be made available at least at district level

The success of the WHO Elimination Strategy suggests that there will gradually be fewer positive smears Leprosy will not however be eradicated by the year 2000 It is therefore essential that there be accessible centres with trained staff who will be able to carry out this procedure accurately As the world tuberculosis situation deteriorates it becomes increasingly necessary to have laboratories in peripheral health units capable of bacterioloshygical examination of sputum Tuberculosis diagnosing centres (possibly one such laboratory per 100000 of the populationo) with an established system of quality control can also be responsible for leprosy microscopy This is easier if the staining technique for both organisms is identicaL From this study it does not appear likely that the choice of decolourizer or its strength is a major factor in the general unreliability of skin smear services in some part of the world

It is possible that this is a much more robust technology than is generally thought This study suggests that the same technique for staining leprosy and tuberculosis bacilli can be used furthermore that acid alcohol is an improvement on sulphuric acid

Acknowledgements

We thank Dr A Colin McDougall of Oxford UK for his advice and encouragement We also

Dccllrier llId ifS Itrellgrh ill rile Z-N StU1I ln

thank the leprosy patients and staff of the BP Kulit Skin Clinic and RS Dayu Leprosy Hospital for their assistance The leprosy control programme of the Indonesian Ministry of

Health in South Sulawesi is supported by the Netherlands Leprosy Relief Association (NSL)

References

I WHO II gllide ttl elilllinting lepros IS a pIIlic heallh problelil WHOILEPlJSI 1st Edn 1995 Leiker DL ~kDollgall AC Teellliutl 8l1id Jilt SIICl1 X(IllIlIIiOIl jor lepros 2nd revised edition Talmi1cp Wurzburl I 1X7

1 DavidsOl AR Deeolorizing of Mrcowcleriulil lepme lilt J [cpr 1943 2 49-51 Davidson AR The tec1mique of staining leprosy hacilli in smears Lepl ReI 19fiO 31 305-307

5 PadmJ Mr- A standard technique of acid fast staining for M leprae in sme~rs Iqn lllliil 1163 35 62-64 ( Ridky llJ amp Ridley OS Stain techniqucs and the morphology of M lepme Lpr Rev 1371 42 RR-95

Ridley 101 Thc cellular eXlldatc-MrcobacleriulIl lepme relationship and the criticll reading of skin Slilears lepr 111 19 60 ~~J-24()

x International Union Against Tuberculosis and Lung Diseases (UATLDI Technical Guide For Sputum Examination For Tuberculosis By Direct Micromiddotopy 1978 In TuJ(fculosis gllid jill 1m illcol1le cOlllllriI 4th eun IUATLD Paris 1996

J WHO l_olior(lon 1((lilliques ill leprosy WHOCDSLEP864 I Hn lI Nilsen T Spare II G Skill lIlIearsC)r leprosy ALERT Addis Ababa DAHWTalmilep 1990 II SLRampTC Skill IIlear ltwllilfllioll Technical Manual Sr No 31 Schicifelin Leprosy Rehabilitation and

Training Cenlre Karigiri India 19R7 J~ Dharlllllldra (cd) -ltprosy Vol I Kothari r1edical Publ Hou~( Bombay I fJ7K u VcttOI11 L PrilZe S Reliahility of skill smear rcsuits ~xpcriences with quality control of skin smears in different I routine services in ieplOs) control programmes Lepr ReI 1189 60 1 g7 -116

Bllku IcuolllltIn PemhcrantltIsan Pcnyakil Kusta (Mallllali workers ill 111e lepros logIYtJIIJlle) Edll IX Department of Health Jakarta 1995

15 Petunjuk Teknis Pcnatalak)anaan Lahoratoriunl Di Pu-kesmas (Technical guidelinesor htalh C(111re ubOrCl(Ory procedurls) Depltlilmcnt of Health Jakltlrta 191)0

16 Kirkwood BR Esset1tials (~lmedi((ll statistics Blackwell Scientific Publications Oxford 1992 17 POflnighalls ltv Diagnosis and management of single lesion leprosy Lepr ReI 1996 67 89-94 18 Desikan KV The risk of relapse after multidrug therapy in leprosy upr Rev 1997 6X 114-116 19 Georgie GD ~lcDollgall AC Skin smears and the bacterial index (Bi) in Illultiple drug therapy leI) contrl)1

programmes an unsatisfactory and potentially hazardous state of affairs 1111 J Iep 1988 56 I () 1middot-1 03 ~o International Union Against Tuberculosis and Lung Diseases (IUATLD) Tuher(ulosis guide for OH incoIJJe

cowllriel 4th cdn IUATLD Paris 1996

131 f)ec(I(lIri~er ([lid its strellgth ill the Z-N staill

The averagc of all thc Bfs using 1 HCI was 2middot31 (with seven negatives) The average HI using 1 HCl was 2A1 (with six negatives) The difference between the BIs wa~ not statistically significant (p lt 0middot2 and gt01) The average BI using 5 H2S04 was 1middot91 (with 11 negatives) Tlw average HI using 25r H2S04 was 2middot21 (with nine ncgatives) Again thc difference between the Bis is not statistically significant (p lt 01 and gtOmiddotOS) When the differences between the BIs ith 1 acid alcohol and 5 sulphuric acid were examined there was an average fall of 0middot41 This was signillcant at the 29c level (p lt 002) suggesting that there might be a real decrease in BI when using sulphuric acid

There was generally good correlation between the four slide results from each patient An exception was founli in patients 15 16 and 17 where the slides stained with 5(1 H2SO were negative whereas the other three were moderately or strongly positive The most likely explanation for this was that these slides were wiped clean on the wrong sick of the slide hence removing all four smears from the slide If the results from these three patients are excluded the average BIs arc 226 and 2middot36 for acid alcohol and 2middot06 and 214 for sulphuric acid The differences between these ligures (betwecn the two acid alcohols the two sulphuric aeids and between I( acid alcohol and Y( sulphuric acid) all fail to re1Ch statistical significance (Ii lt ()) I lt 0middot5 and IJ lt ()middotl respectively)

In 3g of 15~ sites cxamined there was a variation of two or morc BI units (ie Illore tlwn (I

1 O-fold diffelTnce in the number of bacilli) from the same site using differcnt dccolourizers No pattern was obscrved

Thc slides which were stained using an acid alcohol uecolourizer were generally easier to examine than those llsing sulphuric acic Irrespective of concentration the bacilli were sharper clearer more reel the background had accepted more blue for differcntiation and there was little or none of the background pink fuzz that is a feature of the slides which were decolourized llsing Slfc or 257lt H2SO

Discussion

POSITIVITY

This study failed to show that a stronger decolourize resulted in a lower gT The BI was slightly higher with the stronger concentration but not reaching statistical significance (see Figure 1) This was also noted when looking at the results from patients with a low laquo2) BI The Bl was slightly lower with sulphuric acid this reaching significance only when the questionable data from patients 15 16 and 17 were included

POSSIBLE BIAS

All patients were purposefully selected Nu attempt was made to havc a rcpresentative sample of leprosy patients new or otherwise Since the purpose of the study was to comparc results patient by patient analysis using an average BI for all patients per technique is open to question The difference in BI from the same site could also be affected by th~ quality of the smear and within-observer variation Three patients had results which suggest a major laboratory error Analysis was also performcd excluding results from these three patients

DOES IT REALL Y ~ATTER

Most new leprosy patients can be competently diagnosed and commenced on appropriate

132 R de SodellllOi) el ill

Patients

1--1 HCI +3 HCI +5 H2S04 ---25 H2S04l

Figure 1 Comparison of Bl results using four decolourizers

treatment without 1 skin smear However there are some patients who present with single or few lesions but who have early multibacillary disease 17 There are other patients either new or old who have no clearly demonstrable clinical cardinal signs but who have a positive smear There are suspicions that some multi bacillary patients who are released from fixed duration treatment but still with a BI of gt3 may have a significant relapse rate It has been suggsted that regular clinical and bacteriological review after release from treatment is indicated for these patients IS Where communications are difficult it may nut be practical to refer all smear examinations to a I eferral laboratory 19 Hence there is still be a need for skin smear examinatioll to be made available at least at district level

The success of the WHO Elimination Strategy suggests that there will gradually be fewer positive smears Leprosy will not however be eradicated by the year 2000 It is therefore essential that there be accessible centres with trained staff who will be able to carry out this procedure accurately As the world tuberculosis situation deteriorates it becomes increasingly necessary to have laboratories in peripheral health units capable of bacterioloshygical examination of sputum Tuberculosis diagnosing centres (possibly one such laboratory per 100000 of the populationo) with an established system of quality control can also be responsible for leprosy microscopy This is easier if the staining technique for both organisms is identicaL From this study it does not appear likely that the choice of decolourizer or its strength is a major factor in the general unreliability of skin smear services in some part of the world

It is possible that this is a much more robust technology than is generally thought This study suggests that the same technique for staining leprosy and tuberculosis bacilli can be used furthermore that acid alcohol is an improvement on sulphuric acid

Acknowledgements

We thank Dr A Colin McDougall of Oxford UK for his advice and encouragement We also

Dccllrier llId ifS Itrellgrh ill rile Z-N StU1I ln

thank the leprosy patients and staff of the BP Kulit Skin Clinic and RS Dayu Leprosy Hospital for their assistance The leprosy control programme of the Indonesian Ministry of

Health in South Sulawesi is supported by the Netherlands Leprosy Relief Association (NSL)

References

I WHO II gllide ttl elilllinting lepros IS a pIIlic heallh problelil WHOILEPlJSI 1st Edn 1995 Leiker DL ~kDollgall AC Teellliutl 8l1id Jilt SIICl1 X(IllIlIIiOIl jor lepros 2nd revised edition Talmi1cp Wurzburl I 1X7

1 DavidsOl AR Deeolorizing of Mrcowcleriulil lepme lilt J [cpr 1943 2 49-51 Davidson AR The tec1mique of staining leprosy hacilli in smears Lepl ReI 19fiO 31 305-307

5 PadmJ Mr- A standard technique of acid fast staining for M leprae in sme~rs Iqn lllliil 1163 35 62-64 ( Ridky llJ amp Ridley OS Stain techniqucs and the morphology of M lepme Lpr Rev 1371 42 RR-95

Ridley 101 Thc cellular eXlldatc-MrcobacleriulIl lepme relationship and the criticll reading of skin Slilears lepr 111 19 60 ~~J-24()

x International Union Against Tuberculosis and Lung Diseases (UATLDI Technical Guide For Sputum Examination For Tuberculosis By Direct Micromiddotopy 1978 In TuJ(fculosis gllid jill 1m illcol1le cOlllllriI 4th eun IUATLD Paris 1996

J WHO l_olior(lon 1((lilliques ill leprosy WHOCDSLEP864 I Hn lI Nilsen T Spare II G Skill lIlIearsC)r leprosy ALERT Addis Ababa DAHWTalmilep 1990 II SLRampTC Skill IIlear ltwllilfllioll Technical Manual Sr No 31 Schicifelin Leprosy Rehabilitation and

Training Cenlre Karigiri India 19R7 J~ Dharlllllldra (cd) -ltprosy Vol I Kothari r1edical Publ Hou~( Bombay I fJ7K u VcttOI11 L PrilZe S Reliahility of skill smear rcsuits ~xpcriences with quality control of skin smears in different I routine services in ieplOs) control programmes Lepr ReI 1189 60 1 g7 -116

Bllku IcuolllltIn PemhcrantltIsan Pcnyakil Kusta (Mallllali workers ill 111e lepros logIYtJIIJlle) Edll IX Department of Health Jakarta 1995

15 Petunjuk Teknis Pcnatalak)anaan Lahoratoriunl Di Pu-kesmas (Technical guidelinesor htalh C(111re ubOrCl(Ory procedurls) Depltlilmcnt of Health Jakltlrta 191)0

16 Kirkwood BR Esset1tials (~lmedi((ll statistics Blackwell Scientific Publications Oxford 1992 17 POflnighalls ltv Diagnosis and management of single lesion leprosy Lepr ReI 1996 67 89-94 18 Desikan KV The risk of relapse after multidrug therapy in leprosy upr Rev 1997 6X 114-116 19 Georgie GD ~lcDollgall AC Skin smears and the bacterial index (Bi) in Illultiple drug therapy leI) contrl)1

programmes an unsatisfactory and potentially hazardous state of affairs 1111 J Iep 1988 56 I () 1middot-1 03 ~o International Union Against Tuberculosis and Lung Diseases (IUATLD) Tuher(ulosis guide for OH incoIJJe

cowllriel 4th cdn IUATLD Paris 1996

132 R de SodellllOi) el ill

Patients

1--1 HCI +3 HCI +5 H2S04 ---25 H2S04l

Figure 1 Comparison of Bl results using four decolourizers

treatment without 1 skin smear However there are some patients who present with single or few lesions but who have early multibacillary disease 17 There are other patients either new or old who have no clearly demonstrable clinical cardinal signs but who have a positive smear There are suspicions that some multi bacillary patients who are released from fixed duration treatment but still with a BI of gt3 may have a significant relapse rate It has been suggsted that regular clinical and bacteriological review after release from treatment is indicated for these patients IS Where communications are difficult it may nut be practical to refer all smear examinations to a I eferral laboratory 19 Hence there is still be a need for skin smear examinatioll to be made available at least at district level

The success of the WHO Elimination Strategy suggests that there will gradually be fewer positive smears Leprosy will not however be eradicated by the year 2000 It is therefore essential that there be accessible centres with trained staff who will be able to carry out this procedure accurately As the world tuberculosis situation deteriorates it becomes increasingly necessary to have laboratories in peripheral health units capable of bacterioloshygical examination of sputum Tuberculosis diagnosing centres (possibly one such laboratory per 100000 of the populationo) with an established system of quality control can also be responsible for leprosy microscopy This is easier if the staining technique for both organisms is identicaL From this study it does not appear likely that the choice of decolourizer or its strength is a major factor in the general unreliability of skin smear services in some part of the world

It is possible that this is a much more robust technology than is generally thought This study suggests that the same technique for staining leprosy and tuberculosis bacilli can be used furthermore that acid alcohol is an improvement on sulphuric acid

Acknowledgements

We thank Dr A Colin McDougall of Oxford UK for his advice and encouragement We also

Dccllrier llId ifS Itrellgrh ill rile Z-N StU1I ln

thank the leprosy patients and staff of the BP Kulit Skin Clinic and RS Dayu Leprosy Hospital for their assistance The leprosy control programme of the Indonesian Ministry of

Health in South Sulawesi is supported by the Netherlands Leprosy Relief Association (NSL)

References

I WHO II gllide ttl elilllinting lepros IS a pIIlic heallh problelil WHOILEPlJSI 1st Edn 1995 Leiker DL ~kDollgall AC Teellliutl 8l1id Jilt SIICl1 X(IllIlIIiOIl jor lepros 2nd revised edition Talmi1cp Wurzburl I 1X7

1 DavidsOl AR Deeolorizing of Mrcowcleriulil lepme lilt J [cpr 1943 2 49-51 Davidson AR The tec1mique of staining leprosy hacilli in smears Lepl ReI 19fiO 31 305-307

5 PadmJ Mr- A standard technique of acid fast staining for M leprae in sme~rs Iqn lllliil 1163 35 62-64 ( Ridky llJ amp Ridley OS Stain techniqucs and the morphology of M lepme Lpr Rev 1371 42 RR-95

Ridley 101 Thc cellular eXlldatc-MrcobacleriulIl lepme relationship and the criticll reading of skin Slilears lepr 111 19 60 ~~J-24()

x International Union Against Tuberculosis and Lung Diseases (UATLDI Technical Guide For Sputum Examination For Tuberculosis By Direct Micromiddotopy 1978 In TuJ(fculosis gllid jill 1m illcol1le cOlllllriI 4th eun IUATLD Paris 1996

J WHO l_olior(lon 1((lilliques ill leprosy WHOCDSLEP864 I Hn lI Nilsen T Spare II G Skill lIlIearsC)r leprosy ALERT Addis Ababa DAHWTalmilep 1990 II SLRampTC Skill IIlear ltwllilfllioll Technical Manual Sr No 31 Schicifelin Leprosy Rehabilitation and

Training Cenlre Karigiri India 19R7 J~ Dharlllllldra (cd) -ltprosy Vol I Kothari r1edical Publ Hou~( Bombay I fJ7K u VcttOI11 L PrilZe S Reliahility of skill smear rcsuits ~xpcriences with quality control of skin smears in different I routine services in ieplOs) control programmes Lepr ReI 1189 60 1 g7 -116

Bllku IcuolllltIn PemhcrantltIsan Pcnyakil Kusta (Mallllali workers ill 111e lepros logIYtJIIJlle) Edll IX Department of Health Jakarta 1995

15 Petunjuk Teknis Pcnatalak)anaan Lahoratoriunl Di Pu-kesmas (Technical guidelinesor htalh C(111re ubOrCl(Ory procedurls) Depltlilmcnt of Health Jakltlrta 191)0

16 Kirkwood BR Esset1tials (~lmedi((ll statistics Blackwell Scientific Publications Oxford 1992 17 POflnighalls ltv Diagnosis and management of single lesion leprosy Lepr ReI 1996 67 89-94 18 Desikan KV The risk of relapse after multidrug therapy in leprosy upr Rev 1997 6X 114-116 19 Georgie GD ~lcDollgall AC Skin smears and the bacterial index (Bi) in Illultiple drug therapy leI) contrl)1

programmes an unsatisfactory and potentially hazardous state of affairs 1111 J Iep 1988 56 I () 1middot-1 03 ~o International Union Against Tuberculosis and Lung Diseases (IUATLD) Tuher(ulosis guide for OH incoIJJe

cowllriel 4th cdn IUATLD Paris 1996

Dccllrier llId ifS Itrellgrh ill rile Z-N StU1I ln

thank the leprosy patients and staff of the BP Kulit Skin Clinic and RS Dayu Leprosy Hospital for their assistance The leprosy control programme of the Indonesian Ministry of

Health in South Sulawesi is supported by the Netherlands Leprosy Relief Association (NSL)

References

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16 Kirkwood BR Esset1tials (~lmedi((ll statistics Blackwell Scientific Publications Oxford 1992 17 POflnighalls ltv Diagnosis and management of single lesion leprosy Lepr ReI 1996 67 89-94 18 Desikan KV The risk of relapse after multidrug therapy in leprosy upr Rev 1997 6X 114-116 19 Georgie GD ~lcDollgall AC Skin smears and the bacterial index (Bi) in Illultiple drug therapy leI) contrl)1

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